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LC-MS/MS analysis of Prp28-E326BPA experiment

ABSTRACT: We use the BPA crosslinker in Prp28 to “capture” Prp28 in action within the assembling splicing complexes by UV crosslinking. We wish to explore the interacting targets of Prp28-E326BPA within the assembling splicing complexes.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Fu-lung Yeh

LAB HEAD: Tien-Hsien Chang

PROVIDER: PXD024493 | Pride | 2021-04-20

REPOSITORIES: Pride

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			Action	DRS
	20210127-E326-UVn-1.raw	Raw
	20210127-E326-UVn-2.raw	Raw
	20210127-E326-UVp-1.raw	Raw
	20210127-E326-UVp-2.raw	Raw
	ID2mzTab.mzTab.gz	Mztab

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1 - 5 of 25

Publications

Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling.

Yeh Fu-Lung FL Chang Shang-Lin SL Ahmed Golam Rizvee GR Liu Hsin-I HI Tung Luh L Yeh Chung-Shu CS Lanier Leah Stands LS Maeder Corina C Lin Che-Min CM Tsai Shu-Chun SC Hsiao Wan-Yi WY Chang Wei-Hau WH Chang Tien-Hsien TH

Nature communications 20210525 1

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interac ...[more]

PMID: 34035302

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LC-MS/MS analysis of Prp28-K136BPA experiment

Project description:We use the BPA crosslinker in Prp28 to “capture” Prp28 in action within the assembling splicing complexes by UV crosslinking. We wish to explore the interacting targets of Prp28-K136BPA within the assembling splicing complexes.

2021-04-20 | PXD024492 | Pride

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin

Project description:Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR (“Critical sequence”) contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

2023-01-31 | PXD035668 | Pride

A High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid

Project description:In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

2024-06-11 | PXD050493 | Pride

Hsp70 and NAC chaperone systems are in close proximity on the ribosome

Project description:To understand the orientation of Hsp70 Ssb on the ribosome in living Saccharomyces cerevisiae cells we incorporated the noncanonical, photoactivatable amino acid p-benzoyl-L-phenylalanine (Bpa) in place of specific individual endogenous amino acids in Ssb1. To identify the proteins to which Ssb1Bpa crosslinked we performed mass spectrometry. The results revealed that Bpa incorporated at the tip of the “lid” subdomain cross linked to the nascent chain associated complex (NAC), while Bpa incorporated into further down the lid crosslinked to ribosomal protein uL29.

2024-01-08 | PXD046895 | Pride

psiCLIP of SmB, Prp16 and Prp22 in C, C* and P spliceosomal complexes

Project description:Despite the major progress made into spliceosome mechanisms through CryoEM, a major obstacle of this approach is its inability to map the positioning of helicases on the RNA substrate. Here we present a method ‘psiCLIP’ which probes protein-RNA interactions in specific spliceosomal states. The method is based on iCLIP, but is different to previous iCLIP studies in that it is performed on step-specific spliceosomes that are prepared from in vitro splicing extracts, similarly to those prepared for CryoEM. We applied psiCLIP to SmB (C complex), Prp16 (C complex) and Prp22 (C* and P complexes), using pre-mRNA substrates based on UBC4 and ACT1. We also used ATPase deficient dominant negative (dn) mutants of Prp16 and Prp22 to determine the contribution of ATPase activity to binding patterns.

2020-03-15 | E-MTAB-8895 | biostudies-arrayexpress

The Hsp90 molecular chaperone governs client proteins by targeting intrinsically disordered regions

Project description:Molecular chaperones govern proteome health to support cell homeostasis. An essential eukaryotic component of the chaperone system is Hsp90. Using a chemical-biology approach we characterized the features driving the Hsp90 physical interactome.

2023-08-10 | PXD040875 | Pride

Concerted regulation of ISWI by an autoinhibitory domain and the H4 N-terminal tail

Project description:ISWI-family nucleosome remodeling enzymes need the histone H4 N-terminal tail to mobilize nucleosomes. Here we mapped the H4-tail binding pocket of ISWI. Surprisingly the binding site was adjacent to but not overlapping with the docking site of an auto-regulatory motif, AutoN, in the N-terminal region (NTR) of ISWI, indicating that AutoN does not act as a simple pseudosubstrate as suggested previously. Rather, AutoN cooperated with a hitherto uncharacterized motif, termed AcidicN, to confer H4-tail sensitivity and discriminate between DNA and nucleosomes. A third motif in the NTR, ppHSA, was functionally required in vivo and provided structural stability by clamping the NTR to Lobe 2 of the ATPase domain. This configuration is reminiscent of Chd1 even though Chd1 contains an unrelated NTR. Our results shed light on the intricate structural and functional regulation of ISWI by the NTR and uncover surprising parallels with Chd1. Elife. 2017 Jan 21;6. pii: e21477. doi: 10.7554/eLife.21477. [Epub ahead of print]

2017-02-02 | PXD005831 | Pride

Identification of HJURP binding determinants on the Mis18 complex using photo-cross-linking and mass spectrometry

Project description:In the early G1 phase of the cell cycle, human HJURP is recruited to centromeres by the Mis18 complex, which consists of Mis18α, Mis18β and M18BP1. This interaction of HJURP with the Mis18 complex is essential for CENP-A deposition and the preservation of centromere identity. To identify the binding site of HJURP on the Mis18 complex, we used the amber-codon suppression method to incorporate unnatural photo-cross-linking amino acids (Bpa, p-benzoyl-L-phenylalanine; AbK, 3’-azibutyl-N-carbamoyl-lysine) and performed UV-cross-linking and mass spectrometry (MS) experiments.

2019-08-01 | PXD013339 | Pride

Gene expression profiling of Daphnia magna exposed to AgNO3 and AgNP

Project description:Custom D. magna gene expression microarray (Design ID: 023710, Agilent Technologies)were used to characterise gene expression profiles of Daphnia magna neoantes exposed to silver nanoparticles ( AgNPs ) or silver nitrate ( AgNO3 ) for 24 hours.

2016-04-01 | E-MTAB-4041 | biostudies-arrayexpress

Entamoeba histolytica splicing/mRNP proteome

Project description:First, the snRNP component U1A was HA-tagged and the intron of the RabX13 gene was MS2-tagged and amoeba transformats were established with such constructs. Next, amoeba transformants were UV cross-linked and their nucler extracts were immunoprecipitated (IP) with anti-HA agarose or MS2-spharose beads. Precipitates were subjected to tandem mass spectrometry (MS/MS) analyses. MS2-IP helped to discriminate the nuclear roles (chromatin-, co-transcriptional-, splicing-related), of the proteins probed.

2014-09-11 | PXD001080 | Pride

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